RESUMO
Photosymbiodemes are a special case of lichen symbiosis where one lichenized fungus engages in symbiosis with two different photosynthetic partners, a cyanobacterium and a green alga, to develop two distinctly looking photomorphs. We compared gene expression of thallus sectors of the photosymbiodeme-forming lichen Peltigera britannica containing cyanobacterial photobionts with thallus sectors with both green algal and cyanobacterial photobionts and investigated differential gene expression at different temperatures representing mild and putatively stressful conditions. First, we quantified photobiont-mediated differences in fungal gene expression. Second, because of known ecological differences between photomorphs, we investigated symbiont-specific responses in gene expression to temperature increases. Photobiont-mediated differences in fungal gene expression could be identified, with upregulation of distinct biological processes in the different morphs, showing that interaction with specific symbiosis partners profoundly impacts fungal gene expression. Furthermore, high temperatures expectedly led to an upregulation of genes involved in heat shock responses in all organisms in whole transcriptome data and to an increased expression of genes involved in photosynthesis in both photobiont types at 15 and 25°C. The fungus and the cyanobacteria exhibited thermal stress responses already at 15°C, the green algae mainly at 25°C, demonstrating symbiont-specific responses to environmental cues and symbiont-specific ecological optima.
Assuntos
Cianobactérias , Líquens , Líquens/genética , Líquens/microbiologia , Simbiose/genética , Sinais (Psicologia) , Cianobactérias/genética , FilogeniaRESUMO
BACKGROUND: Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution. RESULTS: A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting. CONCLUSIONS: The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.
Assuntos
Ascomicetos/genética , Clorófitas/genética , Líquens/genética , Simbiose/genética , Transferência Genética Horizontal , Genoma FúngicoRESUMO
BACKGROUND: Cyanobacteria of the genus Nostoc are capable of forming symbioses with a wide range of organism, including a diverse assemblage of cyanolichens. Only certain lineages of Nostoc appear to be able to form a close, stable symbiosis, raising the question whether symbiotic competence is determined by specific sets of genes and functionalities. RESULTS: We present the complete genome sequencing, annotation and analysis of two lichen Nostoc strains. Comparison with other Nostoc genomes allowed identification of genes potentially involved in symbioses with a broad range of partners including lichen mycobionts. The presence of additional genes necessary for symbiotic competence is likely reflected in larger genome sizes of symbiotic Nostoc strains. Some of the identified genes are presumably involved in the initial recognition and establishment of the symbiotic association, while others may confer advantage to cyanobionts during cohabitation with a mycobiont in the lichen symbiosis. CONCLUSIONS: Our study presents the first genome sequencing and genome-scale analysis of lichen-associated Nostoc strains. These data provide insight into the molecular nature of the cyanolichen symbiosis and pinpoint candidate genes for further studies aimed at deciphering the genetic mechanisms behind the symbiotic competence of Nostoc. Since many phylogenetic studies have shown that Nostoc is a polyphyletic group that includes several lineages, this work also provides an improved molecular basis for demarcation of a Nostoc clade with symbiotic competence.
Assuntos
Genômica , Líquens/microbiologia , Nostoc/genética , Genoma Bacteriano/genética , Anotação de Sequência Molecular , Nostoc/metabolismo , Nostoc/fisiologia , Organofosfonatos/metabolismo , Análise de Sequência de DNA , SimbioseRESUMO
Bacteria are a major source of natural products that provide rich opportunities for both chemical and biological investigation. Although the vast majority of known bacterial metabolites derive from free-living organisms, increasing evidence supports the widespread existence of chemically prolific bacteria living in symbioses. A strategy based on bioinformatic prediction, symbiont cultivation, isotopic enrichment, and advanced analytics was used to characterize a unique polyketide, nosperin, from a lichen-associated Nostoc sp. cyanobacterium. The biosynthetic gene cluster and the structure of nosperin, determined from 30 µg of compound, are related to those of the pederin group previously known only from nonphotosynthetic bacteria associated with beetles and marine sponges. The presence of this natural product family in such highly dissimilar associations suggests that some bacterial metabolites may be specific to symbioses with eukaryotes and encourages exploration of other symbioses for drug discovery and better understanding of ecological interactions mediated by complex bacterial metabolites.
Assuntos
Vias Biossintéticas/genética , Líquens/genética , Metagenoma/genética , Policetídeo Sintases/genética , Simbiose/genética , Sequência de Bases , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Biologia Computacional , Mineração de Dados , Componentes do Gene , Islândia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metagenômica/métodos , Dados de Sequência Molecular , Estrutura Molecular , Família Multigênica/genética , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Lichens are defined as the specific symbiotic structure comprising a fungus and a green alga and/or cyanobacterium. Up until recently, non-photobiont endothallic bacteria, while known to be present in large numbers, have generally been dismissed as functionally irrelevant cohabitants of the lichen thallus, or even environmental contaminants. Recent analyses of lichen metagenomes and innovative co-culture experiments have uncovered a functionally complex community that appears to contribute to a healthy lichen thallus in several ways. Lichen-associated bacteriomes are typically dominated by several lineages of Proteobacteria, some of which may be specific for lichen species. Recent work has implicated members of these lineages in several important ecophysiological roles. These include nutrient scavenging, including mobilization of iron and phosphate, nitrogen fixation, cellulase, xylanase and amylase activities, and oxidation of recalcitrant compounds, e.g. aromatics and aliphatics. Production of volatile organic compounds, conferring antibacterial and antifungal activity, has also been demonstrated for several lichen-associated isolates. In the present paper we review the nature of non-phototrophic endolichenic bacteria associated with lichens, and give insight into the current state of knowledge on their importance the lichen symbiotic association.
Assuntos
Interações Hospedeiro-Patógeno , Líquens/microbiologia , Proteobactérias/fisiologia , Anti-Infecciosos/metabolismo , Metagenoma , Fixação de Nitrogênio , Simbiose , Compostos Orgânicos Voláteis/metabolismoRESUMO
Although lichens are generally described as mutualistic symbioses of fungi and photosynthetic partners, they also harbour a diverse non-phototrophic microbiota, which is now regarded as a significant part of the symbiosis. However, the role of the non-phototrophic microbiota within the lichen is still poorly known, although possible functions have been suggested, including phosphate solubilization and various lytic activities. In the present study we focus on the bacterial biota associated with the foliose lichen Peltigera membranacea. To address our hypotheses on possible roles of the non-phototrophic microbiota, we used a metagenomic approach. A DNA library of bacterial sequence contigs was constructed from the lichen thallus material and the bacterial microbiota DNA sequence was analysed in terms of phylogenetic diversity and functional gene composition. Analysis of about 30,000 such bacterial contigs from the P. membranacea metagenome revealed significant representation of several genes involved in phosphate solubilization and biopolymer degradation.
Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Bactérias/genética , Líquens/fisiologia , Metagenoma , Fosfatos/metabolismo , Simbiose , Ascomicetos/classificação , Bactérias/classificação , Biologia Computacional , Código de Barras de DNA Taxonômico , Genômica , Dados de Sequência MolecularRESUMO
BACKGROUND: Recent years have witnessed a rising trend in exploring microalgae for valuable carotenoid products as the demand for lutein and many other carotenoids in global markets has increased significantly. In green microalgae lutein is a major carotenoid protecting cellular components from damage incurred by reactive oxygen species under stress conditions. In this study, we investigated the effects of abiotic stressors on lutein accumulation in a strain of the marine microalga D. salina which had been selected for growth under stress conditions of combined blue and red lights by adaptive laboratory evolution. RESULTS: Nitrate concentration, salinity and light quality were selected as three representative influencing factors and their impact on lutein production in batch cultures of D. salina was evaluated using response surface analysis. D. salina was found to be more tolerant to hyper-osmotic stress than to hypo-osmotic stress which caused serious cell damage and death in a high proportion of cells while hyper-osmotic stress increased the average cell size of D. salina only slightly. Two models were developed to explain how lutein productivity depends on the stress factors and for predicting the optimal conditions for lutein productivity. Among the three stress variables for lutein production, stronger interactions were found between nitrate concentration and salinity than between light quality and the other two. The predicted optimal conditions for lutein production were close to the original conditions used for adaptive evolution of D. salina. This suggests that the conditions imposed during adaptive evolution may have selected for the growth optima arrived at. CONCLUSIONS: This study shows that systematic evaluation of the relationship between abiotic environmental stresses and lutein biosynthesis can help to decipher the key parameters in obtaining high levels of lutein productivity in D. salina. This study may benefit future stress-driven adaptive laboratory evolution experiments and a strategy of applying stress in a step-wise manner can be suggested for a rational design of experiments.
Assuntos
Luteína/biossíntese , Microalgas/metabolismo , Estresse Fisiológico , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Carotenoides/metabolismo , Clorofila/metabolismo , Luz , Luteína/química , Microalgas/crescimento & desenvolvimento , Nitratos/química , Nitratos/metabolismo , Pressão OsmóticaRESUMO
There is a particularly high interest to derive carotenoids such as ß-carotene and lutein from higher plants and algae for the global market. It is well known that ß-carotene can be overproduced in the green microalga Dunaliella salina in response to stressful light conditions. However, little is known about the effects of light quality on carotenoid metabolism, e.g., narrow spectrum red light. In this study, we present UPLC-UV-MS data from D. salina consistent with the pathway proposed for carotenoid metabolism in the green microalga Chlamydomonas reinhardtii. We have studied the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield and identified the optimal photon flux for D. salina growth. We found that the major carotenoids changed in parallel to the chlorophyll b content and that red light photon stress alone at high level was not capable of upregulating carotenoid accumulation presumably due to serious photodamage. We have found that combining red LED (75 %) with blue LED (25 %) allowed growth at a higher total photon flux. Additional blue light instead of red light led to increased ß-carotene and lutein accumulation, and the application of long-term iterative stress (adaptive laboratory evolution) yielded strains of D. salina with increased accumulation of carotenoids under combined blue and red light.
Assuntos
Evolução Biológica , Carotenoides/biossíntese , Luz , Volvocida/metabolismo , Volvocida/efeitos da radiação , Biomassa , Biotecnologia/métodos , Cromatografia Líquida , Espectrometria de Massas , Espectrofotometria Ultravioleta , Volvocida/crescimento & desenvolvimentoRESUMO
Lectins are a diverse group of carbohydrate binding proteins often involved in cellular interactions. A lectin gene, lec-2, was identified in the mycobiont of the lichen Peltigera membranacea. Sequencing of lec-2 open reading frames from 21 individual samples showed an unexpectedly high level of polymorphism in the deduced protein (LEC-2), which was sorted into nine haplotypes based on amino acid sequence. Calculations showed that the rates of nonsynonymous versus synonymous nucleotide substitutions deviated significantly from the null hypothesis of neutrality, indicating strong positive selection. Molecular modeling revealed that most amino acid replacements were around the putative carbohydrate-binding pocket, indicating changes in ligand binding. Lectins have been thought to be involved in the recognition of photobiont partners in lichen symbioses, and the hypothesis that positive selection of LEC-2 is driven by variation in the Nostoc photobiont partner was tested by comparing mycobiont LEC-2 haplotypes and photobiont genotypes, as represented by the rbcLX region. It was not possible to pair up the two types of marker sequences without conflicts, suggesting that positive selection of LEC-2 was not due to variation in photobiont partners.
RESUMO
Warming can alter the biogeochemistry and ecology of soils. These alterations can be particularly large in high northern latitude ecosystems, which are experiencing the most intense warming globally. In this meta-analysis, we investigated global trends in how experimental warming is altering the biogeochemistry of the most common limiting nutrient for biological processes in cold ecosystems of high northern latitudes (>50°): nitrogen (N). For comparison, we also analyzed cold ecosystems at intermediate and high southern latitudes. In addition, we examined N-relevant genes and enzymes, and the abundance of belowground organisms. Together, our findings suggest that warming in cold ecosystems increases N mineralization rates and N2 O emissions and does not affect N fixation, at least not in a consistent way across biomes and conditions. Changes in belowground N fluxes caused by warming lead to an accumulation of N in the forms of dissolved organic and root N. These changes seem to be more closely linked to increases in enzyme activity that target relatively labile N sources, than to changes in the abundance of N-relevant genes (e.g., amoA and nosZ). Finally, our analysis suggests that warming in cold ecosystems leads to an increase in plant roots, fungi, and (likely in an indirect way) fungivores, and does not affect the abundance of archaea, bacteria, or bacterivores. In summary, our findings highlight global trends in the ways warming is altering the biogeochemistry and ecology of soils in cold ecosystems, and provide information that can be valuable for prediction of changes and for management of such ecosystems.
Assuntos
Ecossistema , Ciclo do Nitrogênio , Biomassa , Fungos , Nitrogênio , SoloRESUMO
Organisms have evolved different cellular mechanisms to deal with environmental stress, primarily through complex molecular mechanisms including protein refolding and DNA repair. As mutualistic symbioses, lichens offer the possibility of analyzing molecular stress responses in a particularly tight interspecific relationship. We study the widespread cyanolichen Peltigera membranacea, a key player in carbon and nitrogen cycling in terrestrial ecosystems at northern latitudes. We ask whether increased temperature is reflected in mRNA levels of selected damage control genes, and do the response patterns show geographical associations? Using real-time PCR quantification of 38 transcripts, differential expression was demonstrated for nine cyanobacterial and nine fungal stress response genes (plus the fungal symbiosis-related lec2 gene) when the temperature was increased from 5 °C to 15 °C and 25 °C. Principle component analysis (PCA) revealed two gene groups with different response patterns. Whereas a set of cyanobacterial DNA repair genes and the fungal lec2 (PC1 group) showed an expression drop at 15 °C vs. 5 °C, most fungal candidates (PC2 group) showed increased expression at 25 °C vs. 5 °C. PC1 responses also correlated with elevation. The correlated downregulation of lec2 and cyanobacterial DNA repair genes suggests a possible interplay between the symbionts warranting further studies.
Assuntos
Cianobactérias/genética , Fungos/genética , Líquens/microbiologia , Simbiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cianobactérias/classificação , Cianobactérias/isolamento & purificação , Cianobactérias/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/classificação , Fungos/isolamento & purificação , Fungos/fisiologia , Líquens/fisiologia , Filogenia , TemperaturaRESUMO
Like most other lentiviruses, maedi-visna virus (MVV) requires Vif for replication in natural target cells and in vivo. Here, we show that Vif-deficient MVV accumulates G-A mutations in the sequence context characteristic of ovine APOBEC3, consistent with a role of MVV Vif in neutralizing APOBEC3. We studied two point mutations in the vif gene of MVV. One was a tryptophan to arginine mutation that affects the interaction with APOBEC3 and caused G-A hypermutation. The other mutation was a proline to serine mutation that together with a mutation in the capsid protein caused attenuated replication in fetal ovine synovial (FOS) cells but not in sheep choroid plexus (SCP) cells. There was no hypermutation associated with this mutation. These results suggest that MVV Vif exerts more than one function and that there may be interaction between Vif and the capsid. The results also suggest the involvement of an unknown host factor in MVV Vif function.
Assuntos
Produtos do Gene vif/genética , Mutação de Sentido Incorreto , Mutação Puntual , Replicação Viral , Vírus Visna-Maedi/fisiologia , Proteínas do Capsídeo/genética , Fenótipo , Vírus Visna-Maedi/genéticaRESUMO
Mitochondrial genomes from the fungal partners of two terricolous foliose lichen symbioses, Peltigera membranacea and Peltigera malacea, have been determined using metagenomic approaches, including RNA-seq. The roughly 63 kb genomes show all the major features found in other Pezizomycotina, such as unidirectional transcription, 14 conserved protein genes, genes for the two subunit rRNAs and for a set of 26 tRNAs used in translating the 62 amino acid codons. In one of the tRNAs a CAU anticodon is proposed to be modified, via the action of the nuclear-encoded enzyme, tRNA Ile lysidine synthase, so that it recognizes the codon AUA (Ile) instead of AUG (Met). The overall arrangements and sequences of the two circular genomes are similar, the major difference being the inversion and deterioration of a gene encoding a type B DNA polymerase. Both genomes encode the RNA component of RNAse P, a feature seldom found in ascomycetes. The difference in genome size from the minimal ascomycete mitochondrial genomes is largely due to 17 and 20 group I introns, respectively, most associated with homing endonucleases and all found within protein-coding genes and the gene encoding the large subunit rRNA. One new intron insertion point was found, and an unusually small exon of seven nucleotides (nt) was identified and verified by RNA sequencing. Comparative analysis of mitochondrion-encoded proteins places the Peltigera spp., representatives of the class Lecanoromycetes, close to Leotiomycetes, Dothidiomycetes, and Sordariomycetes, in contrast to phylogenies found using nuclear genes.
Assuntos
Ascomicetos/genética , Genoma Mitocondrial , Filogenia , Ascomicetos/classificação , Códon , DNA Fúngico/química , DNA Fúngico/genética , DNA Mitocondrial/química , DNA Mitocondrial/genética , Proteínas Fúngicas/genética , Ordem dos Genes , Metagenoma , Dados de Sequência Molecular , RNA de Transferência/genética , Análise de Sequência de DNA , Sintenia , Transcrição GênicaRESUMO
Lichens and most ascomycete fungi produce polyketide secondary metabolites often with valuable biological activities. Their biosynthesis is primarily governed by large iterative multifunctional type I polyketide synthases. Although there has been good progress studying filamentous non-lichenized fungi, there is limited information on polyketide biosynthesis in lichens and their mycobionts, due to their slow growth, difficulties in establishing pure cultures, and the absence of methods for direct genetic manipulation. However, heterologous expression in a surrogate host offers an alternative approach for exploring lichen polyketide biosynthesis. Here, we report cloning of a type I polyketide synthase gene from the foliose lichen Solorina crocea and its heterologous transcription in the filamentous fungus Aspergillus oryzae, including processing of the transcript. No new polyketide product was detected. The lichen polyketide synthase showed greatest homology with uncharacterized genes from filamentous fungi and lower homology with proteins catalysing biosynthesis of the decaketide alternapyrone and the tetraketide side-chain of squalestatin. The technology platform utilized here presents a useful tool for functional characterization of fungal biosynthetic genes and provides a means for novel production of valuable compounds.
Assuntos
Ascomicetos/enzimologia , Proteínas Fúngicas/genética , Líquens/enzimologia , Policetídeo Sintases/genética , Sequência de Aminoácidos , Ascomicetos/genética , Aspergillus oryzae/genética , Clonagem Molecular , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Líquens/genética , Macrolídeos/química , Macrolídeos/metabolismo , Dados de Sequência Molecular , Policetídeo Sintases/metabolismo , Alinhamento de Sequência , Transcrição GênicaRESUMO
We have shown previously that a type-specific neutralization domain is located within a 39 aa sequence in the fourth variable domain of gp135 in visna/maedi virus. We now show that neutralizing antibodies detected early in infection are directed to this epitope, suggesting an immunodominant nature of this domain. Ten antigenic variants were previously analysed for mutations in this region, and all but one were found to be mutated. To assess the importance of these mutations in replication and neutralization, we reconstructed several of the mutations in an infectious molecular clone and tested the resulting viruses for neutralization phenotype and replication. Mutation of a conserved cysteine was shown to alter the neutralization epitope, whilst the replication kinetics in macrophages were unchanged. Mutations modulating potential glycosylation sites were found in seven of the ten antigenic variants. A frequently occurring mutation, removing a potential glycosylation site, had no effect on its own on the neutralization phenotype of the virus. However, adding an extra potential glycosylation site in the region resulted in antigenic escape. The results indicate that the conserved cysteine plays a role in the structure of the epitope and that glycosylation may shield the principal neutralization site.
Assuntos
Anticorpos Antivirais/imunologia , Cisteína/química , Mutação , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vírus Visna-Maedi/genética , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Células Cultivadas , Plexo Corióideo/citologia , Plexo Corióideo/virologia , Glicosilação , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Dados de Sequência Molecular , Testes de Neutralização , Pneumonia Intersticial Progressiva dos Ovinos/imunologia , Pneumonia Intersticial Progressiva dos Ovinos/virologia , Ovinos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Vírus Visna-Maedi/imunologiaRESUMO
Polyketides are a group of natural products that have gained much interest due to their use as antibiotics, cholesterol lowering agents, immunosuppressors, and as other drugs. Many organisms that naturally produce polyketides are difficult to cultivate and only produce these metabolites in small amounts. It is therefore of general interest to transfer polyketide synthase (PKS) genes from their natural sources into heterologous hosts that can over-produce the corresponding polyketides. In this study we demonstrate the heterologous expression of 6-methylsalicylic acid synthase (6-MSAS), naturally produced by Penicillium patulum, in the yeast Saccharomyces cerevisiae. In order to activate the PKS a 4'-phosphopantetheinyl transferase (PPTase) is required. We therefore co-expressed PPTases encoded by either sfp from Bacillus subtilis or by npgA from Aspergillus nidulans. The different strains were grown in batch cultures. Growth and product concentration were measured and kinetic parameters were calculated. It was shown that both PPTases could be efficiently used for activation of PKS's in yeast as good yields of 6-MSA were obtained with both enzymes.
Assuntos
Policetídeo Sintases/biossíntese , Saccharomyces cerevisiae/enzimologia , Salicilatos/metabolismo , Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biomassa , Reatores Biológicos/microbiologia , Técnicas de Cultura de Células , Ativação Enzimática , Fermentação , Cinética , Plasmídeos , Saccharomyces cerevisiae/genética , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Transformação GenéticaRESUMO
Maedi-visna virus (MVV) is a lentivirus of sheep causing chronic inflammatory disease of the lungs (maedi) and the nervous system (visna). We have previously shown that a duplicated sequence in the long terminal repeat (LTR) of MVV is a determinant of cell tropism. Here, we demonstrate that deletion of a CAAAT sequence from either one of the repeats resulted in poor virus growth in sheep choroid plexus cells. A duplication in the LTR encompassing the CAAAT sequence was found in four neurological field cases that were sequenced, but no duplication was present in the LTRs from seven maedi cases; one maedi isolate was mixed. These results indicate that the duplication in the LTR is associated with neurovirulence.
Assuntos
Plexo Corióideo/virologia , Sequências Repetidas Terminais/genética , Vírus Visna-Maedi/fisiologia , Vírus Visna-Maedi/patogenicidade , Animais , Sequência de Bases , Células Cultivadas , Plexo Corióideo/citologia , Dados de Sequência Molecular , Pneumonia Intersticial Progressiva dos Ovinos/virologia , Deleção de Sequência , Carneiro Doméstico , Visna/virologia , Vírus Visna-Maedi/genética , Vírus Visna-Maedi/isolamento & purificaçãoRESUMO
Maedi-visna virus (MVV) is a lentivirus of sheep sharing several key features with the primate lentiviruses. The virus causes slowly progressive diseases, mainly in the lungs and the central nervous system of sheep. Here, we investigate the molecular basis for the differential growth phenotypes of two MVV isolates. One of the isolates, KV1772, replicates well in a number of cell lines and is highly pathogenic in sheep. The second isolate, KS1, no longer grows on macrophages or causes disease. The two virus isolates differ by 129 nucleotide substitutions and two deletions of 3 and 15 nucleotides in the env gene. To determine the molecular nature of the lesions responsible for the restrictive growth phenotype, chimeric viruses were constructed and used to map the phenotype. An L120R mutation in the CA domain, together with a P205S mutation in Vif (but neither alone), could fully convert KV1772 to the restrictive growth phenotype. These results suggest a functional interaction between CA and Vif in MVV replication, a property that may relate to the innate antiretroviral defense mechanisms in sheep.
Assuntos
Produtos do Gene vif/fisiologia , Macrófagos/virologia , Vírus Visna-Maedi/fisiologia , Animais , DNA Viral/análise , Produtos do Gene vif/genética , Genoma Viral , Mutação , Ovinos , Doenças dos Ovinos/virologia , Replicação Viral , Vírus Visna-Maedi/patogenicidadeRESUMO
Four sheep were infected intratracheally with an attenuated molecular clone of maedi-visna virus (MVV). All four became infected. Ten months later these sheep were challenged intratracheally with a genetically similar but pathogenic clone of MVV. Four unvaccinated sheep were infected simultaneously. All sheep became infected by the challenge virus. The vaccinated sheep were not protected against superinfection with the challenge clone. However, virus was isolated more frequently from the blood of the unvaccinated controls than of the vaccinated animals and ten times more frequently from lungs of unvaccinated sheep than from lungs of vaccinated sheep at sacrifice, indicating partial protection.
Assuntos
Imunidade nas Mucosas/imunologia , Vacinas Virais/imunologia , Vírus Visna-Maedi/imunologia , Visna/imunologia , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/biossíntese , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Feminino , Hibridização In Situ , Ovinos , Superinfecção/prevenção & controle , Vacinas Atenuadas/imunologia , Carga Viral , Visna/prevenção & controle , Visna/virologia , Vírus Visna-Maedi/isolamento & purificaçãoRESUMO
We have investigated the role of vif in maedi-visna virus (MVV), a lentivirus of sheep, by studying in vitro replication of vif-deleted MVV in several cell types, and the effects of vif deletion on in vivo infection. By measuring RT activity, we found that in comparison to wild-type MVV, growth of vif-deleted MVV was similar in fetal ovine synovial (FOS) cells, highly attenuated in sheep choroid plexus (SCP) cells, and not detectable in macrophages, natural target cells of MVV. Productive infection by vif-deleted MVV could not be demonstrated in sheep. An increased mutation frequency was observed in DNA produced by endogenous reverse transcription of viral RNA in vif-deleted virions, indicating the existence of a factor comparable in action to human APOBEC3G. These results suggest that the vif gene of MVV is essential for infectivity and that the Vif protein protects the viral genome from enpackaged mutagenic activities.